Tanaproget derivatives, metabolites, and uses thereof

ABSTRACT

A method of generating synthetic metabolites of tanaproget derivatives thereof is provided. These compounds and methods of using these derivatives for detecting tanaproget metabolites in samples are provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit under 35 USC 119(e) of U.S. PatentApplication No. 60/601,254, filed Aug. 13, 2004.

BACKGROUND OF THE INVENTION

The present invention provides novel derivatives of tanaproget.

Tanaproget is a potent, non-steroidal progesterone receptor agonistbeing developed for use in contraception as an alternative to currentlyavailable oral contraceptives. The elimination of steroid progestinsfrom contraceptive regimens may reduce the common side effects of oralcontraceptives.

SUMMARY OF INVENTION

The present invention provides metabolites of the active compoundtanaproget. These compounds are useful in methods and kits formonitoring therapy with tanaproget.

Among these metabolites, a rare S-linked glucuronide conjugate,S-glucuronide tanaproget has been isolated and now synthesized. Havingfirst been identified as a metabolite, when a tanaproget glucuronide isdelivered to a subject, it is a prodrug that is enzymatically cleaved totanaproget by glucuronidase in vivo. Thus, the invention providestanaproget glucuronide derivatives formulated for administration as atanaproget prodrug. In one embodiment, administration is by the oralroute to maximize the advantages of glucuronidase activity in the gut.

Other aspects and advantages of the invention will be apparent from thefollowing detailed description of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The derivatives of the invention are unique synthetic metabolites thatare believed to be bioequivalent to the metabolites produced by asubject following administration of tanaproget. Thus, the derivatives ofthe invention are useful as standards in kits for monitoring tanaprogettherapy, and for generating antibodies specific for tanaprogetmetabolites. Such antibodies are useful for monitoring and studying theeffects of tanaproget therapy.

Further, the tanaproget glucuronide derivatives of the invention can bedelivered to a subject as a pro-drug, which is cleaved to the activeform of tanaproget in vivo. Thus, the invention further providespharmaceutical compositions and kits containing the tanaprogetglucuronide derivatives of the invention, and methods of using same todeliver tanaproget to a subject. A subject may include any mammal,preferably female, including humans and non-humans.

As used herein, the PR agonist compound termed “NSP-989” or “tanaproget”[Wyeth] is characterized by a core structure:

Methods for the synthesis of the illustrated core structure,5-(4,4-dimethyl-2-thioxo-1,4-dihydro-2H-3,1-benzoxazin-6-yl)-1-methyl-1H-pyrrole-2-carbonitrile,are described in U.S. Pat. No. 6,436,929, U.S. patent application Ser.No. 11/113,794 (filed Apr. 25, 2005), and U.S. Provisional PatentApplication Nos. 60/675,550 (filed Apr. 28, 2005); 60/675,551 (filedApr. 28, 2005); 60/675,599 (filed Apr. 28, 2005); 60/675,737 (filed Apr.28, 2005); and 60/675,738 (filed Apr. 28, 2005), as are uses for thiscompound. Other suitable synthetic methods for obtaining tanaproget willbe readily apparent to one of skill in the art. The present invention isnot limited by the means for producing tanaproget.

The present invention provides glucuronide derivatives of the above corestructure. The derivatives of the invention may contain one or moreasymmetric centers and may thus give rise to optical isomers anddiastereoisomers. While shown without respect to stereochemistry below,the present invention includes such optical isomers anddiastereoisomers; as well as the racemic and resolved, enantiomericallypure R and S stereoisomers; as well as other mixtures of the R and Sstereoisomers and pharmaceutically acceptable salts thereof.

In one embodiment, the glucuronide moiety is attached through the N atomin the oxindole ring. This derivative can be characterized by thestructure:

In another embodiment, the glucuronide moiety is attached through the Satom bound to the oxindole ring. In one embodiment, this derivative ischaracterized by the structure:

In other embodiments, these compounds have the followingstereochemistry.

The tanaproget glucuronide derivatives of the invention may be producedsynthetically, using conventional techniques. For example, a solution oftanaproget in anhydrous neutral solvent (e.g., DMF) is added dropwiseunder a nitrogen atmosphere to a solution of a strong base (e.g., sodiumhydride) diluted in the solvent and then cooled using dry ice. Aftermixing, a solution of acetobromo-α-D-glucuronic acid ester is added. Thereaction solution is then warmed to room temperature, and stirred. Afterabout 8 to 24 hours, the reaction solution is partitioned between waterand an organic solvent, e.g., ethyl acetate. The aqueous layer isextracted. The combined organic layers are washed with saturated sodiumchloride (NaCl) solution (100 mL), dried, and the solvent removed invacuo.

Alternatively, the glucuronide derivates of the invention can beproduced in an enzymatic system using suitable methods.

Conventional techniques can be used to recover the purified crudetanaproget derivatives. In one embodiment, the tanaproget derivativesmay be purified by the means described in U.S. Provisional PatentApplication No. 60/675,738 (filed Apr. 28, 2005), hereby incorporated byreference. In another embodiment, the crude extract can be passedthrough an HPLC reverse phase column with a gradient of solvents toremove unreacted tanaproget and the starting materials and reagents fromthe crude products. Suitable solvents for use in the gradients of acolumn can be readily selected by one of skill in the art. In theexample herein, acetonitrile/methanol and acetonitrile/ammonium acetatewere used as the solvents. The fractions, which contain tanaprogetderivatives, are combined and the purified solvents are evaporated toprovide the tanaproget derivative mixture. Thin layer chromatography orother chromatographic methods known in the art may be used forpurification.

In order to isolate the individual derivatives, the purified mixture canbe subjected to further separation using chromatographic techniques. Forexample, high performance liquid chromatography (HPLC) can be used.Suitable columns and conditions for separation will be readily apparentto one of skill in the art given the present disclosure.

In another aspect, this invention includes pharmaceutical compositionsand treatments which comprise administering to a subject (e.g., a femaleof child bearing age for contraception or another mammal for therapeuticpurposes) a pharmaceutically effective amount of one or more glucuronidederivatives of tanaproget as described above as agonists of theprogesterone receptor.

The tanaproget glucuronide compounds of this invention, used alone or incombination, can be utilized in methods of contraception,pre-menopausal, peri-menopausal and/or post-menopausal hormonereplacement therapy, and the treatment and/or prevention of skindisorders, dysfunctional bleeding, estrus synchronization, uterineleiomyomata, endometriosis, polycystic ovary syndrome, and carcinomasand adenocarcinomas of the endometrium, ovary, breast, colon, andprostate. Additional uses of the invention include stimulation of foodintake.

The term “skin” is meant to describe the outer covering of a mammalianform including, without limitation, the epidermis, dermis, andsubcutaneous tissues. Typically, the skin can include other componentssuch as hair follicles and sweat glands. Skin disorders include, e.g.,acne and hirsutism.

The term “acne” is meant to include any skin disorder where a skin porebecomes blocked and/or thereby becomes inflamed. The term acne includeswithout limitation superficial acne, including comedones, inflamedpapules, superficial cysts, and pustules; and deep acne, including deepinflamed modules and pus-filled cysts. Specific acne conditions caninclude, but are not limited to, acne vulgaris, acne comedo, papularacne, premenstrual acne, preadolescent acne, acne venenata, acnecosmetica, pomade acne, acne detergicans, acne excoriee, gram negativeacne, acne rosacea, pseudofolliculitis barbae, folliculitis, perioraldermatitis, and hiddradenitis suppurativa. The term “hirsutism” is meantto describe a skin disorder where an overgrowth of hair growth isobserved in areas of the body which are not normally subject toexcessive hair growth.

A number of skin disorders can be treated with the compounds of thepresent invention, including skin disorders of the hair follicles andsebaceous glands. In one embodiment, skin disorders such as acne andhirsutism, among others, can be treated according to the presentinvention.

Other skin disorders including dry/chapped skin, seboria, psoriasis, oralopecia can be treated using the compounds and compositions of theinvention. The invention is also useful for treating the skin againstthe effects of environmental conditions.

This invention also includes pharmaceutical compositions utilizing thecompounds herein, optionally in combination with a pharmaceuticallyacceptable carrier or excipient. When the compounds are employed for theabove utilities, they may be combined with one or more pharmaceuticallyacceptable carriers or excipients, for example, solvents, diluents andthe like, and may be administered orally in such forms as tablets,capsules, dispersible powders, granules, suspensions containing, forexample, from about 0.05 to 5% of suspending agent, syrups containing,for example, from about 10 to 50% of sugar, and elixirs containing, forexample, from about 20 to 50% ethanol, and the like, or parenterally inthe form of sterile injectable solutions or suspensions containing fromabout 0.05 to 5% suspending agent in an isotonic medium. Suchpharmaceutical preparations may contain, for example, from about 25 toabout 90% of the tanaproget derivative in combination with the carrier,more usually between about 5% and 60% by weight.

The effective dosage of tanaproget derivative employed may varydepending on the particular tanaproget glucuronide derivative employed,the mode of administration and the severity of the condition beingtreated. However, in general, satisfactory results are obtained when thecompounds of the invention are administered at a daily dosage of fromabout 0.5 to about 500 mg/kg of animal body weight, optionally given individed doses one to four times a day, or in a sustained release form.For most large mammals, the total daily dosage is from about 1 to 100mg, or from about 2 to 80 mg. Dosage forms suitable for internal usecomprise from about 0.5 to 500 mg of the tanaproget derivative inintimate admixture with a solid or liquid pharmaceutically acceptablecarrier. This dosage regimen may be adjusted to provide the optimaltherapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

These tanaproget glucuronide derivatives may be administered orally aswell as by intravenous, intramuscular, or subcutaneous routes. Solidcarriers include starch, lactose, dicalcium phosphate, microcrystallinecellulose, sucrose and kaolin, while liquid carriers include sterilewater, polyethylene glycols, non-ionic surfactants and edible oils suchas corn, peanut and sesame oils, as are appropriate to the nature of thetanaproget derivative and the particular form of administration desired.Adjuvants customarily employed in the preparation of pharmaceuticalcompositions may be advantageously included, such as flavoring agents,coloring agents, preserving agents, and antioxidants, for example,vitamin E, ascorbic acid, BHT and BHA.

The preferred pharmaceutical compositions from the standpoint of ease ofpreparation and administration are solid compositions, particularlytablets and hard-filled or liquid-filled capsules. Oral administrationof the tanaproget glucuronide derivatives is presently preferred.

These tanaproget derivatives may also be administered parenterally orintraperitoneally. Solutions or suspensions of these tanaprogetderivatives as a free base or pharmacologically acceptable salt can beprepared in water suitably mixed with a surfactant such ashydroxypropylcellulose. Dispersions can also be prepared in glycerol,liquid, polyethylene glycols and mixtures thereof in oils. Underordinary conditions of storage and use, these preparations contain apreservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringe ability exits. It must be stable underconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacterial and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol (e.g., glycerol, propylene glycol and liquid polyethyleneglycol), suitable mixtures thereof, and vegetable oil.

The present invention provides kits or packages of pharmaceuticalformulations designed for use in regimens described herein. In oneembodiment, these kits are designed for daily oral delivery over 21-day,28-day, 30-day, or 31-day cycles, among others, and for one oraldelivery per day. When the compositions are to be deliveredcontinuously, a package or kit can include the composition in eachtablet. When the compositions are to be delivered with periodicdiscontinuation, a package or kit can include placebos on those dayswhen the composition is not delivered.

In one embodiment, the kits are organized to indicate a single oralformulation or combination of oral formulations to be taken on each dayof the cycle, including oral tablets to be taken on each of the daysspecified, and in a further embodiment, one oral tablet will containeach of the combined daily dosages indicated.

In one embodiment, a kit can include a single phase of a daily dosage ofthe compound of the invention over a 21-day, 28-day, 30-day, or 31-daycycle. Alternatively, a kit can include a single phase of a daily dosageof the compound of the invention over the first 21 days of a 28-day,30-day, or 31-day cycle. A kit can also include a single phase of adaily dosage of the compound of the invention over the first 28 days ofa 30-day or 31-day cycle.

In a further embodiment, a kit can include a single combined phase of adaily dosage of the compound of the invention and an estrogen over a21-day, 28-day, 30-day, or 31-day cycle. Alternatively, a kit caninclude a single combined phase of a daily dosage of the compound of theinvention and an estrogen over the first 21 days of a 28-day, 30-day, or31-day cycle. A kit can also include a single combined phase of a dailydosage of the compound of the invention and an estrogen over the first28 days of a 30-day or 31-day cycle.

In another embodiment, a 28-day kit can include a first phase of from 14to 28 daily dosage units of the compound of the invention; a secondphase of from 1 to 11 daily dosage units of an estrogen; and,optionally, a third phase of an orally and pharmaceutically acceptableplacebo for the remaining days of the cycle.

In yet a further embodiment, a 28-day kit can include a first phase offrom 14 to 21 daily dosage units of the compound of the invention; asecond phase of from 1 to 11 daily dosage units of an estrogen; and,optionally, a third phase of an orally and pharmaceutically acceptableplacebo for the remaining days of the cycle. In another embodiment, a28-day kit can include a first phase of from 18 to 21 daily dosage unitsof a compound of the invention; a second phase of from 1 to 7 dailydosage units of an estrogen; and, optionally, an orally andpharmaceutically acceptable placebo for each of the remaining 0 to 9days in the 28-day cycle.

In another embodiment, a 28-day kit can include a first phase of 21daily dosage units of a compound of the invention; a second phase of 3daily dosage units for days 22 to 24 of an estrogen; and, optionally, athird phase of 4 daily dosage units of an orally and pharmaceuticallyacceptable placebo for each of days 25 to 28.

In still another embodiment, the daily dosage of each pharmaceuticallyactive component of the regimen remains fixed in each particular phasein which it is delivered. It is further preferable that the daily doseunits described are to be delivered in the order described, with thefirst phase followed in order by the second and third phases. To helpfacilitate compliance with each regimen, the kits may also contain theplacebo described for the final days of the cycle.

A number of packages or kits are known in the art for the use indispensing pharmaceutical agents for oral use. In one embodiment, thepackage has indicators for each day of the 28-day cycle, and may be alabeled blister package, dial dispenser package, or bottle.

Metabolites and Uses Thereof

The tanaproget glucuronide derivatives of the invention are useful formonitoring therapy with tanaproget or with a tanaproget prodrug (e.g., aS-glucuronide tanaproget compound) in a subject. Additionally, theinvention provides other tanaproget metabolites useful for monitoringtherapy with tanaproget and its prodrugs. When used as a reagent and/ora standard, the tanaproget metabolite compounds may be labeled, e.g.,with a radioactive, fluorescent, or calorimetric tag.

In one embodiment, the invention further provides an isolated tanaprogetmetabolite, which can be enzymatically or synthetically produced. Such ametabolite can be selected from among a tanaproget glucuronidederivative. Other suitable metabolites contain the tanaproget corestructure and optional substitutions, including, e.g., tanaproget havinga sulfate moiety located on the thiocarbonyl group; tanaproget having ahydroxy group located on the pyrrole ring; tanaproget having a hydroxygroup located on the phenylpyrrole ring; and tanaproget having acarbamate in place of the thiocarbonyl group.

One or more of these tanaproget metabolites can serve as a standard,i.e., for comparison purposes, in a method for detecting the presence ofa tanaproget metabolite in a sample. A “sample” as used herein refers toa biological sample, such as, for example, tissue or fluid isolated froman individual (including without limitation plasma, serum, cerebrospinalfluid, urine, lymph, tears, saliva and tissue sections) or from in vitrocell culture constituents, as well as samples from the environment.

In another embodiment, one or more of the tanaproget metabolites can beused to generate an antibody or antibodies that are used to detect thepresence of the tanaproget metabolites in a sample. Suitably, theantibody is a monoclonal or polyclonal antibody specific for atanaproget derivative. In one desirable embodiment, such an antibodyselectively binds to the tanaproget derivative of the invention, anddistinguishes that metabolite from tanaproget and other metabolitesthereof.

The term “antibody” as used herein is intended to include fragmentsthereof which are specifically reactive with tanaproget and/or itsmetabolites, e.g., an Fv fragment and a F(ab)2 fragment.

An antibody specific to a tanaproget derivative of the invention can beprepared using standard techniques wherein the antigen is a derivativeof the invention. See, e.g., Sambrook, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.

The polyclonal and monoclonal antibodies to specific sites of atanaproget metabolite may be used for development of immunoassays ortherapeutic drug monitoring (TDM) kits. Such assays could include, butare not limited to, direct, inhibition, competitive or sandwichimmunoassays (ELISA or other assay systems), RIA, solid or liquid phaseassays or automated assay systems.

Where a competitive assay is used, the competitor for the antibody maybe a tanaproget derivative of the invention bound to the assay plate, ora labeled derivative, e.g., a fluorolabeled derivative, a radiolabeledderivative, or a tritiated derivative.

Where desired, a kit can be used to facilitate the methods of theinvention.

A kit of the invention may contain an appropriately labeled tracer, anantibody, standard, instructions for use, and packaging. The label forthe tracer may be any suitable label, e.g., a radioactive, fluorescentor calorimetric label. Where convenient, the components of the kit maybe in lyophilized form.

The assay procedure of the invention has the advantages that it may becarried out rapidly and simply using standard bioanalytical equipment togive accurate and reproducible results. Also, whole blood may be usedwithout the need for extraction.

The invention also provides an assay kit suitable for detecting theamount of tanaproget metabolite in a sample (e.g., blood or urine). Inone embodiment, the kit comprises a binding competitor that displacesthe pharmaceutical from tanaproget metabolite in the sample; and anantibody that binds to the pharmaceutical but not significantly to thebinding competitor.

The following examples are provided to illustrate the invention and donot limit the scope thereof. One skilled in the art will appreciate thatalthough specific reagents and conditions are outlined in the followingexamples, modifications can be made which are meant to be encompassed bythe spirit and scope of the invention.

The following examples are illustrative of the methods for generatingcompounds of the invention.

EXAMPLE 1 Preparation of Tanaproget S-Glucuronide from Rat LiverMicrosomes

The glucuronide of tanaproget was prepared in male rat liver microsomes,and the structure was identified as a S-glucuronide conjugate by liquidchromatography (LC)/mass spectrometry (MS) and nuclear magneticresonance (NMR) spectroscopy. This metabolite was also identified as amajor metabolite in both male and female rat, dog, and human livermicrosomes, and it was also the major drug related component found inrat, dog, and human plasma. This S-glucuronide can also be syntheticallyprepared simultaneously with a N-glucuronide. The two syntheticglucuronides could be separated by HPLC and their structures were allcharacterized by LC/MS and NMR spectroscopy. In the following scheme,the NSP-989 terminology is used in the place of tanaproget.

A. Incubation of Tanaproget and Extraction of Tanaproget Glucuronide

Liver microsomes from Sprague-Dawley rats were prepared in-house using adifferential ultracentrifugation method described by Lake [Lake, B. InBiochemical Toxicology: A Practical Approach, Snell, K, Mullock, B(eds), IRL Press: England, 1987, 183-215] with slight modifications.Microsomal protein and cytochrome P450 content were determined by themethod of Bradford [Bradford, M M Anal. Biochem. 1976; 72:248-254] andOmura and Sato [Omura, T, Sato, R J. Biol. Chem. 1964; 238:2370-2378],respectively. The protein concentration and P450 content were 50.9 mg/mLand 0.42 nmol/mg protein, respectively. Ammonium acetate, magnesiumchloride, and uridine diphosphoglucuronic acid (UDPGA) were purchasedfrom Sigma Chemical Company (St. Louis, Mo.). The solvents used forextraction and for chromatographic analysis were HPLC grade or ACSReagent Grade (Mallinckrodt Baker, Phillipsburg, N.J.).

Incubations (100 mL) were performed with tanaproget (40 μM), UDPGA (5mM), magnesium chloride (10 mM), and male rat liver microsomes (1.5mg/mL), in 0.1 M potassium phosphate buffer, pH 7.4 at 37° C. Thesamples were pre-incubated for 1 minute at 37° C., and the reactionswere initiated by the addition of UDPGA. The sample was cooled using anice bath to stop the reaction after 3 hours. Unreacted tanaproget wasremoved from the samples by two extractions with diethyl ether (200mL×2).

Tanaproget glucuronide and remaining unreacted tanaproget were extractedby solid phase extraction using C-18 cartridges and elution withmethanol (10 mL). The methanol eluent was dried by rotary evaporationunder vacuum at room temperature. The residues were extracted with 50%acetonitrile in water (5 mL) and centrifuged at 3500 rpm for 15 min.Aliquots (800 μL) of supernatants were analyzed by a Waters 2690® HPLCsystem with a semi-preparative column. Separation of the tanaprogetmetabolites was accomplished on a Phenomenex Luna™ column (C18, 250×10mm ID, 5 μm particle size) (Phenomenex, Torrance, Calif.) andmetabolites were detected by monitoring UV absorbance at 310 nm. Theautosampler temperature was set to 6° C., while the column was at roomtemperature. Ammonium acetate (10 mM, pH 4.5) and acetonitrile were usedas mobile phase A and B, respectively. The following gradient at a flowrate of 2 mL/min with a 5 min post run re-equilibration was employed: 0min 20% B, 1 min 20% B, 10 min 40% B, 20 min 70% B, 25 min 95% B, 28 min95% B, 30 min 20% B. Under these conditions, the tanaproget glucuronidepeak (M1) at 15.5 min was collected for NMR spectroscopic analysis.Fractions containing the glucuronide conjugate peak were collected intoclean tubes and frozen on dry ice immediately after collection. Allglucuronide fractions were combined and acetonitrile was removed byrotary evaporation. The glucuronide was extracted from the aqueouseluates by solid phase extraction using C-18 cartridges. Water (2 mL)was used to wash residual buffer and 50% methanol in water was used toelute the glucuronide conjugate. The methanol/water eluates, were driedby rotary evaporation under vacuum at room temperature. The remainingaqueous solutions were transferred to a 5 mL conical vial to removewater by lyophilization. Drying of the solid residue was continuedovernight to remove additional moisture prior to NMR spectroscopicanalysis.

B. HPLC/MS Analysis Conditions:

A Micromass Quattro Ultima™ triple quadrupole mass spectrometer (WatersCorp., Milford, Mass.) was used in this work. It was equipped withelectrospray ionization (ESI) interface and operated in both thepositive and negative ionization modes. Settings for the massspectrometer were: ESI spray 2.5 KV, cone 50 V, mass resolution 0.7Da±0.2 Da width at half height, desolvation gas flow 900-1000 L/h, conegas flow 50-80 L/h, source block temperature 80° C., desolvation gastemperature 250° C. LC/MS data were analyzed with Micromass MassLynxsoftware (Waters Corp., versions 3.5 and 4.0).

Solvents used for chromatographic analysis of tanaproget glucuronideisolated from rat liver microsomes were HPLC grade or ACS Reagent Grade(Mallinkrodt Baker, Phillipsburg, N.J. and EMD Chemicals, Gibbstown,N.J.). The HPLC system in conjunction with the mass spectrometer was aWaters Alliance model 2695™ HPLC system. It was equipped with a built-inautosampler and a model 996 diode array UV detector set to monitor210-350 nm. Separations were accomplished on a Phenomenex Luna C18(2)column (150×2 mm, 5 μm) (Phenomenex, Torrance, Calif.) with a Deltabond™C18 guard column (10×2 mm) (ThermoElectron Corp., Bellefonte, Pa.). Theflow rate was 0.3 mL/min. During LC/MS sample analysis, up to 10 min ofthe initial flow was diverted away from the mass spectrometer prior toevaluation of metabolites. Mobile phase A was 10 mM ammonium acetate inwater, pH 4.5, (diluted from a 0.5 M stock solution of equal molaramounts of ammonium acetate and acetic acid) and mobile phase B wasacetonitrile. The linear mobile phase gradient used was: 0 min 10% B, 1min 10% B, 10 min 15% B, 35 min 17.5% B, 36 min 25% B, 50 min 30% B, 55min 50% B, 60 min 90% B, 62 min 90% B, 65 min 10% B, 75 min 10% B.

C. LC/MS Results

Tanaproget glucuronide produced protonated and deprotonated molecularions ([M+H]⁺and [M−H]⁻, at m/z 474 and 472, respectively), whichindicated a molecular weight of 473. This was 176 Da larger thantanaproget. Loss of 176 Da from m/z 474 in the positive ionization massspectrum and from m/z 472 in the negative ionization mass spectrumgenerated the fragment ions at m/z 298 in the positive ionization modeand m/z 296 in the negative ionization mode, which are assigned astanaproget. The glucuronic acid ion fragment was observed at m/z 175 innegative ionization mode. The m/z 339 fragment ion appears to be fromfragmentation of the glucuronic acid ring. These data were consistentwith glucuronic acid conjugation of tanaproget.

D. NMR Spectroscopy

Deuterated dimethylsulfoxide (DMSO-d₆) was used for all NMR samples. Forthe metabolite samples isolated from rat liver microsomes, dissolutionwas done in a glove bag under argon to reduce absorption of atmosphericwater. Typically 50 μL aliquots of a total of 200 μL of DMSO-d₆ wereused to rinse the vial containing the vacuum dried sample and thentransferred into a 3 mm NMR tube. NMR spectra were obtained at 500(Varian Inova™ instrument), and 600 (Bruker Avance™ instrument) MHz. Thebulk of the experimental NMR work was performed on the Varian Inova 500™MHz instrument equipped with a Varian™ 3 mm ¹H observe indirectdetection probe. Chemical shifts δ (ppm) are reported relative tointernal TMS (δ0.0) for ¹H and ¹³C. In DMSO-d₆, ¹H chemical shifts arereferenced to residual protonated DMSO at δ2.49, and ¹³C chemical shiftsare referenced to internal DMSO-d₆ at δ39.5. Chemical shifts for ¹⁵N arereported relative to liquid ammonia (δ0.0) and referenced to externalformamide at δ112.0. Proton multiplicities are reported as s=singlet,d=doublet, dd=doublet of doublets, and m=multiplet. J values are givenas ¹H-¹H coupling constants in Hz.

General parameters for ¹H-NMR experiments include a 5000 Hz spectralwidth, 32K data points, a 45° pulse width, a 1 second relaxation delayand the averaging of from 32 to >1000 scans, depending on sampleconcentration. Line broadening (˜0.5 Hz) or gaussian processing routineswere used to increase the signal-to-noise (S/N). General parameters fora ¹³C-NMR experiment include a 25,000 Hz spectral width, 64K datapoints, a 45° pulse width, a 1 second relaxation delay and averaging ofat least 10,000 scans to achieve optimal S/N. In addition, 2 Hz linebroadening was applied to increase S/N. All spectra were acquired at 25°C.

Several types of 2D NMR experiments were utilized to determine ¹H-¹H and¹H-¹³C connectivities. These included a gCOSY experiment for thedetermination of three-bond ¹H-¹H connectivities, gHSQC and gHMBCexperiments for the determination of one-, two-, three-, and four-bond¹H-¹³C-connectivities, and a NOESY or ROESY experiment for thedetermination of through-space connectivities.

Despite several attempts at using freshly isolated tanaprogetglucuronide samples, the purity and concentration of the samples weretypically low (<70% pure and an estimated total amount of <20 μg insolution) which hampered acquisition of complete 2D NMR datasets. Inaddition, during the process of the microsomal incubation and subsequentisolation steps, the solution tended to rapidly produce the carbamateanalog of tanaproget (the sulfur of tanaproget replaced by oxygen).Nevertheless, sufficient chemical shift, coupling, and 2D NMRcorrelations were obtained to make nearly complete ¹H and ¹³Cassignments of tanaproget glucuronide isolated from rat livermicrosomes. However, the location of the glucuronide attachment from themicrosomally derived sample alone could not be determined. Only one keyproton-carbon heteronuclear correlation was observed in the gHMBC NMRspectrum of the rat liver microsome incubation sample, that from H-1′(5.10 ppm) to C-6 (161.63 ppm). However, this same crosspeak wasexpected whether the metabolite was an N-or an S-glucuronide (i.e.3-bond coupling in either case).

EXAMPLE 2 Preparation of Synthetic Tanaproget Glucuronide Conjugates

A. HPLC/MS and NMR Analysis Conditions:

HPLC/MS data for the synthetic compounds were acquired using a WatersAlliance 2695™ HPLC coupled to a Waters ZQ™ mass spectrometer. Ingeneral, the samples were analyzed using an open-access LC/MS method aspreviously described [Mallis, L M, Sarkahian, A B, Kulishoff, J M, Jr.,Watts, W L, Jr. J. Mass Spectrom. 2002; 37:889-896].

Deuterated dimethylsulfoxide (DMSO-d₆) was used for all NMR samplesexcept for 2, which was dissolved in CDCl₃ (deuterated solvents fromAldrich, Milwaukee, Wis.). NMR spectra were obtained at 300 (Bruker DPX™instrument), 400 (Varian Inova™ instrument), 500 (Varian Inova™instrument), and 600 (Bruker Avance™ instrument) MHz. The bulk of theexperimental NMR work was performed on the Varian Inova 500™ MHzinstrument equipped with a Varian™ 3 mm ¹H observe indirect detectionprobe. Chemical shifts δ (ppm) in DMSO-d₆ are reported as describedpreviously for tanaproget glucuronide. In CDCl₃, ¹H chemical shifts arereferenced to residual protonated CHCl₃ at δ7.27, and ¹³C chemicalshifts are referenced to internal CDCl₃ at δ77.7. Chemical shifts for¹⁵N are reported as described previously for tanaproget glucuronide.

General parameters for ¹H-NMR and ¹³C-NMR experiments are as describedpreviously for tanaproget glucuronide. All spectra were acquired at 25°C. 2D NMR experiments used to determine ¹H-¹H and ¹H-¹³C connectivitieswere gCOSY, gHSQC, gHMBC, NOESY and ROESY.

B. Preparation of the Protected S-Glucuronide Acid (2) of Tanaproget

A solution of tanaproget (0.292 g, 1.0 mmol) in anhydrous dimethylformamide (DMF) (10 mL) was added dropwise under a nitrogen atmosphereto a solution of sodium hydride (NaH) (0.052 g, 2.2 mmol) in DMF (50 mL)that was cooled to approximately −70° C. (dry ice). After stirring for10 min, a solution of acetobromo-α-D-Glucuronic acid methyl ester (0.396g, 1 mmol) in DMF (10 mL) was added dropwise. The reaction solution wasthen warmed to room temperature, and stirred for a total of 24 hours.After 24 hours, the reaction solution was partitioned between water (100mL) and ethyl acetate (100 mL). The aqueous layer was extracted againwith ethyl acetate (100 mL). The combined organic layers were washedwith saturated sodium chloride (NaCl) solution (100 mL), dried(magnesium sulfate, MgSO₄), and the solvent removed in vacuo. Aliquotsof the crude material were chromatographed under low-pressurereversed-phase C18 (RediSep/ISCO, 125×25 mm ID, 40 μm particle size)conditions with a gradient of 50-95% acetonitrile/water. Fractionseluting with 75-80% acetonitrile/water contained the S-protected(D)-Glucuronic acid derivative (2) of tanaproget. A total ofapproximately 200 mg of (2) was obtained at 95% purity based on NMRspectroscopic analysis (32% isolated yield). All chemicals werepurchased from Aldrich (Milwaukee, Wis.) and used without furtherpurification or drying.

C. Preparation of N-(3) and S-Glucuronic Acid (4) Derivatives ofTanaproget

In a separate reaction, a solution of tanaproget (0.146 g, 0.5 mmol) inanhydrous DMF (5 mL) was added dropwise under a nitrogen atmosphere to asolution of NaH (0.027 g, 1.1 mmol) in DMF (25 mL) that was cooled toapproximately −70° C. (dry ice). After stirring for 10 min, a solutionof acetobromo-α-D-glucuronic acid methyl ester (0.198 g, 0.5 mmol) inDMF (5 mL) was then added dropwise. The reaction solution was thenwarmed to room temperature, and stirred for a total of 8 hours. Thereaction solution was partitioned between water (100 mL) and ethylacetate (100 mL). The aqueous layer was extracted again with ethylacetate (100 mL). The combined organic layers were washed with saturatedNaCl solution (100 mL), dried (MgSO₄), and solvent removed in vacuo toyield 0.350 g of crude reaction material. To a portion of this material(0.210 g) was added a solution of MeOH/Hunig's base ((iso-Pr)₂NEt)/H₂O(5 mL/2 mL/2 mL) and the solution was stirred for 8.5 hours at roomtemperature. The pH of the reaction solution was then adjusted to 2.5using HCl (concentrated, approximately 1.5 mL) and chromatographed bysemi-preparative reversed-phase HPLC using YMC-Pack CN 150×20 mm, S-5 μmcolumn with a gradient of 15-35% acetonitrile in 10 mM (aqueous)ammonium acetate. From repeated injections, approximately 5 mg of theN-(D)-glucuronic acid derivative (3) (3% isolated yield from tanaproget)and 15 mg of the S-(D)-glucuronic acid derivative (4) (10% isolatedyield from tanaproget) was purified to >98% purity for NMR analysis andLC/MS analysis and comparison.

EXAMPLE 3 Comparison of Synthetic Compounds to M1

From extensive comparison of the spectral and chromatographic data ofthe microsomally-derived metabolite and the synthetic compounds, themetabolite has been determined to be the S-(β)-D-glucuronide oftanaproget.

The positive ionization mode LC/MS spectrum (not shown) of syntheticcompound 2, the protected S-glucuronide of tanaproget, gave a protonatedmolecular ion [M+H]⁺ at 614, indicating a molecular weight of 613. Lossof 297 Da gave nt/z 317, which is assigned to [M+H−tanaproget]⁺. An ionsignal at m/z 257 is assigned to m/z 317—acetic acid (C₂H₄O₂), and anobserved ion at m/z 197 is assigned to m/z 257—acetic acid (C₂H₄O₂).Also, an ion at m/z 155 is assigned to m/z 197—acetyl (C₂H₃O)+H. Theprotonated and deprotonated LC/MS spectra (not shown) of theS-glucuronide 4 gave an [M+H]⁺ ion at 474 and an [M−H]⁻ ion at m/z 472,respectively, (as was also the case for 3, spectra not shown),indicating glucuronidation of tanaproget. Also observed for 4 in thepositive and negative ionization mode mass spectra are the ions m/z 298and m/z 296, assigned as tanaproget, that is, the loss of glucuronicacid. In addition, a fragment at m/z 175, assigned as glucuronic acid,was also detected in the negative ionization spectrum.

HPLC comparisons were also made to confirm whether the major syntheticglucuronide of tanaproget, S-glucuronide 4, was identical to tanaprogetglucuronide, now proposed to be the S-glucuronide from the NMR and massspectral results described above. Five different types of reverse phaseHPLC columns were used under two different mobile phase conditions.These ten HPLC conditions covered a wide range of selectivity asevidenced by the change of elution order of the major and minorcomponents in the samples. To compare and match the retention time ofthe major peaks in the two samples, spiking experiments were performed.The synthetic glucuronide that was determined to be S-glucuronide 4 wasfound to have identical retention times as the tanaproget glucuronidemetabolite under all ten HPLC conditions.

Key NMR correlations in the synthetic compounds 3 and 4 which locatedthe site of glucuronidation in tanaproget are described below. Theproton chemical shift and coupling constant for the anomeric proton(H-1′) with β-stereochemistry on the N-glucuronide is 6.31 ppm, and is adoublet with a coupling constant of 9.5 Hz. The carbon chemical shift ofthe anomeric carbon (C-1′) is 90.2 ppm. The carbon chemical shift of thebenzoxazine-2-thione carbon (C-6) of the N-glucuronidated metabolite oftanaproget is 188.0 ppm; this compared with the thiocarbonyl carbon ofthe benzoxazine-2-thione group observed in the parent molecule,tanaproget, at 182.8 ppm.

There are four important 3-bond correlations observed in the HMBCspectrum (not shown) of this molecule. They are from the anomeric proton(H-1′) to the benzoxazine-2-thione carbon (C-6) and to the sp² aromaticcarbon at 131.5 ppm (C-4), and from the protons H-7 and H-9, observed at7.50 ppm and 7.57 ppm, respectively, to C-4. The proton chemical shiftof the proton (H-10) that is in a position peri- to the N-glucuronidehas moved downfield to 7.80 ppm, compared to 7.13 ppm in the parentmolecule, indicating the proximity of the glucuronic acid group (andpossibly the carboxylic acid moiety) to this proton. ¹H—¹⁵N gHMBCexperiments were run on all compounds studied, but signals from the keynitrogen N-5 were not observed for any compound except tanaproget(δ144.81), which had an HMBC crosspeak to H-10. The other nitrogenexpected, N-15, was only observed in tanaproget (δ154.99) and in 3(δ155.40), and in each compound had gHMBC correlations to H-17, H-18,and H-19.

The proton chemical shift and coupling constant for the anomeric proton(H-1′) with β-stereochemistry on the synthetic S-glucuronide 4 is 5.11ppm, and is a doublet with a coupling constant of 10.2 Hz. The carbonchemical shift of the anomeric carbon (C-1′) is 85.0 ppm. The carbonchemical shift of the derivatized benzoxazine-2-thione carbon (C-6) ofthis S-glucuronidated metabolite of tanaproget is observed at 161.3 ppm,quite upfield shifted from the observed thiocarbonyl carbon chemicalshift (182.8 ppm) of the benzoxazine-2-thione group observed in theparent molecule, tanaproget. There is a 3-bond correlation observed inthe HMBC spectrum of this molecule between the anomeric proton (H-1′) ofthe β-glucuronic acid and the derivatized benzoxazine-2-thione carbon(C-6). Finally, there is a 2-bond correlation observed in the HMBCspectrum between the gem-dimethyl protons (H-11 and H-12) observed at1.59 ppm and 1.70 ppm and the carbon (C-2) to which this group isattached, observed at 81.5 ppm. These data confirm that thebenzoxazine-2-thione (N(C═S)O) group of tanaproget did not rearrange toa thiolcarbamate (N(C═O)S) group before S-glucuronidation.

Comparison of the ¹H NMR spectra and the NMR data derived fromtanaproget glucuronide and the two synthetic compounds 3 and 4, revealedthat tanaproget glucuronide must be the S-glucuronide derivative oftanaproget. The H1′ chemical shift for tanaproget glucuronide is 5.10ppm, and 5.11 ppm for 4, compared to H-1′ at 6.31 ppm observed for 3.Also, C-6, in the benzoxazine-2-thione part of tanaproget, resonates at˜161 ppm for both tanaproget glucuronide and 4, but at 188.0 ppm for 3and at 182.8 ppm for tanaproget.

EXAMPLE 4 Enzymatic Hydrolysis of Tanaproget Glucuronide ConjugateMetabolite

In order to confirm that that the tanaproget glucuronide derivative is aprodrug when delivered to patients, the ability of the glucuronideconjugate to be enzymatically cleaved by a glucuronidase which is nativeto the gastrointestinal tract in humans was used in the following assay.

Pooled urine samples (4-8 hr) from healthy women were hydrolyzed byGlusulase®. Aliquots (1 mL) of pooled urine were adjusted to pH 5 with0.5 mL of 0.6 M sodium acetate buffer. The diluted urine was mixed withGlusulase® (9,000 units/mL, 100 μL) and incubated at 37° C. for 1 hrwith gentle shaking. The reaction was stopped by the addition of 2 mL ofacetone and the precipitate was removed by centrifugation. Thesupernatant was dried under nitrogen in a TurboVap™ (Caliper LifeSciences, Hopkinton, Mass.). The residues were reconstituted with 1 mLof 60% methanol in water and subsequently analyzed by HPLC and LC/MS,which confirmed the conversion of glucuronide into parent drugtanaproget. Control incubations were conducted under the sameconditions, but without adding Glusulase®, or with Glusulase® and 10 mMof saccharolactone (β-glucuronidase inhibitor) and no conversion ofglucuronide conjugate into tanaproget was observed.

EXAMPLE 5 Pharmacology

Tanaproget glucuronides are a prodrug of tanaproget, a first-in-classnon-steroidal progesterone receptor agonist for use primarily incontraception. The effect of tanaproget glucuronides on alkalinephosphase activity in T47D cells is analyzed as follows.

A. Reagents:

Culture medium: DMEM:F12 (1:1) (GIBCO, BRL) supplemented with 5% (v/v)charcoal stripped fetal bovine serum (not heat-inactivated), 100 U/mLpenicillin, 100 μg/mL streptomycin, and 2 mM GlutaMax (GIBCO, BRL).

Alkaline phosphatase assay buffer: I. 0.1M Tris-HCl, pH 9.8, containing0.2% Triton X-100, 0.1M Tris-HCl, pH 9.8, containing 4 mM p-nitrophenylphosphate (Sigma).

B. Cell Culture and Treatment:

Frozen T47D cells are thawed in a 37° C. water bath and diluted to280,000 cells/mL in culture medium. To each well in a 96-well plate(Falcon, Becton Dickinson Labware), 180 μl of diluted cell suspension isadded.

Twenty μl of reference or test compounds diluted in the culture mediumis then added to each well. The cells are incubated at 37° C. in a 5%CO₂ humidified atmosphere for 24 hours. For high throughput screening,one concentration of each compound will be tested at 0.3 μg/mL. Based onan average molecular weight of 300 g/mol for the compounds in thelibrary, the concentration is approximately 1 μM. Subsequently, activecompounds will be tested in dose response assays to determine EC50.

C. Alkaline Phosphatase Enzyme Assay:

At the end of treatment, the medium is removed from the plate. Fifty μlof assay buffer I is added to each well. The plates are shaken in atiter plate shaker for 15 min. Then 150 μl of assay buffer II is addedto each well. Optical density measurements are taken at 5 min intervalsfor 30 min at a test wavelength of 405 nM.

D. Analysis of Dose-response Data.

For reference and test compounds, a dose response curve is generated fordose vs. the rate of enzyme reaction (slope). Square root-transformeddata are used for analysis of variance and nonlinear dose response curvefitting for both agonist and antagonist modes. Huber weighting is usedto down-weight the effects of outliers. EC₅₀ values are calculated fromthe retransformed values. JMP software (SAS Institute, Inc.) is used forboth one-way analysis of variance and non-4 linear dose responseanalysis in both single dose and dose response studies.

E. Results

Tanaproget S-glucuronide had 0.1 nM with 60% efficacy as compared toprogesterone.

Tanaproget can be regenerated by enzymatic hydrolysis experiments, suchas described in Example 4 above.

EXAMPLE 6 Additional Tanaproget Metabolites

Using the methods described in Example 1 for obtaining the tanaprogetglucuronide metabolites, additional tanaproget metabolites were observedin the male rat liver microsomal preparations and in male and femalemonkey liver microsome preparations using known techniques.

Metabolite M2 was observed in male rat liver microsomal preparations.This metabolite produced a [M−H]—at m/z 312. The product ion at m/z 58from NCS-, indicating an unchanged thioamide group, was also observedfor NSP-989. Product ions at m/z 159 and 195 indicated the pyrrole ringas the site of metabolism. Therefore, metabolite M2 was proposed to be ahydroxy-NSP-989 with the hydroxy group at the pyrrole moiety.

Metabolite M3 was observed in male rat and male and female monkey livermicrosome preparations. This metabolite produced a [M−H]—at m/z 312. Theproduct ion at m/z 58 from NCS-, indicating an unchanged thioamidegroup, was also observed for NSP-989. The product ion at m/z 237,observed for NSP-989 at m/z 220, indicated oxidation of either thephenyl or pyrrole ring. Product ions at m/z 195 and 252 were consistentwith oxidation of the phenyl or pyrrole ring. Therefore, metabolite M3was proposed to be a hydroxy-NSP-989 with the hydroxy group at thephenyl or pyrrole moiety.

Metabolite M4 was observed in all in vitro metabolism samples. Thismetabolite produced a [M−H]—at m/z 280, which was 16 atomic mass units(amu) less than NSP-989. The lack of a product ion at m/z 58 and the 16amu shift in molecular weight indicated a modified thioamide group.Product ions, observed at m/z 129, 220 and 234 were also present forNSP-989. Metabolite M4 also had the same HPLC retention time and production spectrum (data not shown) as synthetic NSP-989-carbamate. Therefore,metabolite M4 was identified as NSP-989-carbamate.

Metabolite M6 was observed in dog and in rat liver microsomepreparations. This metabolite produced a [M−H]—at m/z 344. The productions at m/z 80 and 81 indicated the presence of a sulfate group. Productions at m/z 220 and 234 indicated that the non-thiocarbonyl portions ofthe NSP-989 molecule were unchanged. Therefore, metabolite M6 wasidentified as NSP-989 sulfate(6-(5-Cyano-1-methyl-1H-pyrrol-2-yl)-4,4-dimethyl-4H-benzo[d][1,3]oxazine-2-sulfonicacid).

These metabolites can be purified from microsome preparations usingmethods described above for the glucuronide derivatives. Alternatively,these metabolites can be generated using convention synthetictechniques.

All patent, patent publications, and other publications listed in thisspecification are incorporated herein by reference. While the inventionhas been described with reference to particular embodiments, it will beappreciated that modifications can be made without departing from thespirit of the invention. Such modifications are intended to fall withinthe scope of the appended claims.

1. An isolated and purified S-glucuronide of tanaproget characterized bythe structure:


2. A synthetic S-glucuronide of tanaproget characterized by thestructure:


3. The synthetic S-glucuronide of tanaproget of claim 2 having a purityof greater than about 98%.
 4. The synthetic S-glucuronide of tanaprogetof claim 2 prepared by reacting tanaproget, sodium hydride, andacetobromo-α-D-glucuronide acid methyl ester.
 5. The syntheticS-glucuronide of tanaproget of claim 2 prepared by: (a) reactingtanaproget, sodium hydride, and acetobromo-α-D-glucuronide acid methylester; (b) reacting the product of step (a) with a solution of methanoland Hunig's base; and (c) acidifying the product of step (b) usinghydrochloric acid.
 6. The synthetic S-glucuronide of tanaproget of claim5, further prepared by: (d) separating the synthetic S-glucuronide oftanaproget by chromatography.
 7. A composition comprising apharmaceutically acceptable carrier and a S-glucuronide of tanaprogetaccording to claim
 2. 8. A method for preparing a syntheticS-glucuronide of tanaproget characterized by the structure:

said method comprising reacting tanaproget, sodium hydride, andacetobromo-α-D-glucuronide acid methyl ester.
 9. A method for preparinga synthetic S-glucuronide of tanaproget characterized by the structure:

said method comprising: (a) reacting tanaproget, sodium hydride, andacetobromo-α-D-glucuronide acid methyl ester; (b) reacting the productof step (a) with a solution of methanol and Hunig's base; and (c)acidifying the product of step (b) using hydrochloric acid.
 10. Themethod according to claim 9, further comprising: (d) separating thesynthetic S-glucuronide of tanaproget using chromatography.